Our first study concerning the antimicrobial activities of squalamine 1
demonstrated its efficiency towards fungal and bacterial strains with Minimum Inhibitory Concentrations (MIC) varying from 2.5 to 25 µg/mL (–). It is also noteworthy that similar activities have been demonstrated against sensitive and resistant Gram-negative bacteria (Escherichia coli
and Pseudomonas aeruginosa
). The re-use of “old” drugs such as polymyxins has been proposed as an alternative or rescue therapy for patient infected by MDR strains.
We have recently reported that two clinical Enterobacter aerogenes
isolates have developed resistance to polymyxins involving an alteration of LPS after colistin was used during the therapy. This modification did not alter the protein profile of outer membrane.
The first isolate, strain C () presenting a polymyxin B susceptibility was sensitive to low concentrations of squalamine 1
. Interestingly, clinical isolates D and E that presented a high level of polymyxin resistance (32-fold increase of MIC) exhibited a decrease of squalamine susceptibility with a five-fold increase of the corresponding MIC. This result suggested that the alterations of LPS previously reported in these isolates and causing the resistance towards polymyxin B
, are able to modulate the squalamine activity. In this context, regarding the other antibiotic families, squalamine offers advantages associated with its activity properties. The squalamine action is preserved even in MDR clinical isolates that overexpress various mechanisms of resistance including drug efflux pumps, alteration of membrane permeability caused by absence of porins, enzymatic barrier, all well-known mechanisms which induce high level of resistance towards quinolones, ß-lactams, phenicols, etc (–). For instance: (i) strain 289 was completely devoid of porins, expressed high level of AcrAB-Tol C efflux and a simultaneous overproduction of β-lactamase activity, (ii) strain 298 (289 derivative) exhibited the same phenotype but was deleted of Tol C efflux component, (iii) strain C was porin defficient, overexpressed AcrAB-Tol C efflux and exhibited a lipopolysaccharide (LPS) wild type profile, (iv) strains D and E had same phenotype plus LPS modifications.
Thus, the activity of squalamine 1
suggests in a first approach that its biological activity results from the synergistic combination of an anionic bile salt with spermidine, each of which independently exhibit considerably less antibiotic activity than squalamine 1
Antimicrobial activities of squalamine 1.
Antimicrobial activities of squalamine 1 towards bacteria resistant strains.
Antibacterial susceptibility of various Multidrug resistant (MDR) E. aerogenes clinical isolates expressing various antibiotic resistance mechanisms.
Even if strong antibacterial activities have been noticed, the mechanism of action of squalamine towards Gram-negative bacteria remains questionable. Thus, two possible modes of action for such an antibacterial molecule can be underlined (i) competitive binding to a cell-surface exposed receptor (e.g.
such as porin)
involved in key cellular processes and (ii) channel or pore formation in the cytoplasmic membrane. Recently, Katsu et al.
examined the structure-activity relationship between original polyamines (naphthylacetylspermine and methoctramine) and the outer membrane of Gram-negative bacteria demonstrating that lipophilic moieties and a number of amino groups in polyamines were important to permeabilisation.
A bioluminescence method was used to determine the effect of squalamine addition on the intracellular pool of bacterial ATP. The detection of external concentration of ATP was then used as a reporter reflecting the permeabilizing effect of squalamine along with the dose-effect relationships. Thus, it clearly appears that for a squalamine concentration of about 20 µg/mL, 80% of the intracellular ATP has been released in the medium suggesting the disruption of the membrane barrier (). In addition to ATP release measurements, effect of squalamine on bacterial membrane integrity was also assessed using the cell-impermeable DNA/RNA dye propidium iodide (PI) (). Results showed that squalamine caused a dose-dependent increase in PI-associated fluorescence. At a 1.25 µg/mL concentration, squalamine did not significantly affect PI-associated fluorescence (2±3.5-fold increase, p
0.42). Increase only started to be significant at 2.5 µg/mL (8.0±3.4 fold increase (p
<0.05)) and was maximal at 25 µg/mL squalamine concentration (110.0±12.5-fold (p
<0.001)). Similarly, CTAB known to cause bacterial permeabilisation, induced equivalent increases in PI-associated fluorescence 100.0±11.5-fold increase compared to vehicle-treated bacteria, p
<0.001). Finally, in order to investigate membrane alterations associated with squalamine action, we have used an assay based on fluorescent microscopy (Live/Dead BacLight, Molecular Probes) (). In that assay, live bacteria appear green whereas dead bacteria with an alteration of their membrane permeability are red. At 0 or 1.25 µg/mL of squalamine, all bacteria appeared green (). Red/dead bacteria started to be observed with concentration of squalamine higher than 2.5 µg/mL (). Moreover, the cytotoxicity increased when bacteria colonies are treated with a higher squalamine concentration (i.e. 100 µg/mL), under this condition, all the bacteria were stained red () in contrast to the untreated ones fluorescing green.
Measurement of squalamine concentration effect on E. coli ATP efflux.
Effect of squalamine on bacterial membrane integrity assessed by fluorescence measurement of propidium iodide – DNA/RNA interactions.
Fluorescence-based microscopic evaluation of the effect of squalamine on bacterial integrity and survival.
On the other hand, it is well known that the effects of chaotropic agents can be impaired by exogenous divalent cationic ions (Mg2+
In this context, the effect of various monovalent or divalent ions on bactericidal activity of squalamine 1
has been investigated using 1 mM concentration salts. As shown in the addition of monovalent salts did not block the activity of squalamine towards E. coli
whereas this later was completely abolished by the same concentration of divalent ions such as MgCl2
. Regarding these divalent salts, it was demonstrated that the full activity of squalamine is obtained at low concentrations i.e. approximately 0.09 mM.
Effects of various monovalent or divalent salt solutions (1 mM) on bactericidal activity of squalamine (2.5 µg/mL).
Moreover, using bioluminescence method, a noticeable inhibition of the E. coli ATP efflux squalamine-dependent was observed in the presence of divalent ions at various concentrations after 10 minutes of incubation (). Thus, NaCl and NaH2PO4 did not lead to any inhibition on E. coli ATP efflux in the presence of squalamine whereas a dramatic inhibition of this efflux was observed in the presence of CaCl2 or MgCl2. Moreover, a total inhibition of the ATP efflux was reached by a concentration of about 5 mM or 2.5 mM for MgCl2 or CaCl2, respectively.
ATP efflux inhibition in E. coli in the presence of squalamine (5 µg/mL) and various mono and divalent salt solutions.
Many groups have observed the antibacterial effect of cationic surfaces on Gram-positive as well as Gram-negative bacteria. This suggests that the mechanism is not system-specific, contrary to that which is generally the case with antibiotics. We surmise, as already hinted by others
, that the death process involves electrostatic interactions and is related to the high density of charges exposed at the surface of bacterial membranes. The architecture of LPS, the main component of outer leaflet of the outer membrane, favors the presence of a large number of negative charges that may stimulate the interactions with cationic substrates.
The role of LPS is partially suggested with the modulation of squalamine activity in polymyxin resistant isolates and ().
To propose the existence of a charge-density threshold in the squalamine mode of action, we are helped by recent advances in the understanding of the electrostatic interactions between polyelectrolyte chains and oppositely charged surfaces.
It has been gradually realized that adsorption in such cases is driven by the release in solution of the counterions initially confined within the respective electrical double layers. The same process applies to bacteria, which can be crudely considered as large two dimensional polyelectrolytes. Upon adsorption on a cationic solid substrate, the electrostatic compensation of the negative charges of the bacterial envelope is provided by the cationic charges of the substrate, and the bacteria lose their natural counterions.
As previously outlined, squalamine is an amphipathic compound which interacts with various membrane glycerophospholipids with distinct affinities.
As phosphatidylglycerol is the main glycerophospholipid in bacterial membranes whereas phosphatidylcholine is more abundant in eukaryotic membranes, this may explain why squalamine could kill bacteria more easily than mammalian cells. Nevertheless, although Gram-positive bacteria have a single membrane that is enriched in phosphatidylglycerol, Gram-negative bacteria also have an external membrane in which the predominant lipid is lipopolysaccharide (LPS). LPS is the major glycolipid recovered from a Folch extract of Gram-negative E. coli
bacteria. To study the potential interaction with squalamine and a reconstituted bacterial membrane containing LPS, a lipid extract of E. coli
enriched in LPS was spread at the air-water interface where it formed a stable lipid monolayer. Squalamine was then added in the aqueous subphase and its insertion within the LPS film was assessed by surface pressure measurements
As shown in , squalamine penetrated the LPS monolayer at concentrations as low as 0.5 µg/mL. In contrast, higher doses of squalamine were necessary to allow its insertion in monolayers consisting of either neutral glycosphingolipids or gangliosides extracted from lymphocytes. Thus, as far as glycolipids are concerned in early squalamine-membrane interactions, it is clear that bacterial LPS is significantly more active than eucaryotic glycolipids. Squalamine also interacted with matured lipid A (the membrane-anchored backbone of LPS), in a divalent cation-dependent way. Indeed, this squalamine-membrane interaction is highly cationic divalent ion dependent which is consistent with the previously demonstrated lack of activity of squalamine in the presence of such ions in the medium. Moreover, squalamine interacted very poorly with GalCer, but very actively with ceramide (Cer), the membrane-anchored backbone of sphingolipids. This may suggest that the insertion of squalamine into eukaryotic membranes could be impaired by the sugar part of glycolipids. Overall these data provide a biochemical basis for the potent activity of squalamine on bacterial Gram-negative and Gram-positive membranes and its relative lack of activity on eukaryotic membranes. Further physicochemical studies will be conducted in the near future in order to decipher the molecular mechanisms (including divalent cation dependence) controlling this striking lipid selectivity.
Measurements of squalamine interactions with various bacterial and eukaryotic lipids.
Squalamine is a membrane-active molecule that targets the membrane integrity as demonstrated by the ATP release and dye entry. Consequently, its activity may depend on the membrane lipid composition. It is worthwhile mentioning that the alteration of LPS involved in the polymyxin-resistant clinical isolates moderately changes the squalamine MIC preserving the activity spectrum of the molecule compared to polymyxin B. Thus, if we consider that squalamine acts as a “membranotropic” molecule, it remains possible to observe less susceptible strains like those isolated after polymyxin treatment. However, the resistant variants must preserve a sensitivity level since the adaptation stress requires strong changes in membrane structure which drastically deal with intrinsic membrane stability and the bacterial fitness. In addition, this molecule shows a preserved activity against bacterial pathogens presenting a noticeable MDR phenotype concerning usual antibiotics. Squalamine has membranotropic properties regarding its bacterial membrane activity and due to its structure containing a cholestanol core it exhibits a moderate level of side effect on eukaryotic cells at doses that kill MDR bacterial pathogens. In this context and because of its structure, action and its relative insensitivity to efflux resistance mechanisms, squalamine may be an alternate way to combat MDR pathogens and by pass the gap regarding the failure of new active antibacterial molecules. This aspect is especially important since some recently described molecules having an active antibacterial spectrum are also substrates for efflux pump systems resulting in a decrease of activity in MDR strains, e.g.
peptide deformylase inhibitor, plectasin, platensimycin.